The subunit analogous to the d-subunit of ATP synthase from bovine heart mitochondria was isolated from the purified yeast enzyme. Partial protein sequences were determined by direct methods. From this information, two oligonucleotide probes were constructed and used for screening a DNA genomic bank of Saccharomyces cerevisiae. The sequence of yeast subunit d was deduced from the DNA sequence of ATP7 gene. Mature yeast subunit d is 173 amino acids long. Its NH2-terminal serine is blocked by an N-acetyl group, and the protein has no processed NH2-terminal sequence other than the removal of the initiator methionine. The protein is predominantly hydrophilic. The amino acid sequence is 22% identical and 44% homologous to bovine subunit d. A null mutant was constructed. The mutant strain was unable to grow on glycerol medium. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity, and the catalytic sector F1 was loosely bound to the membranous part. The mutant mitochondria did not contain subunit d, and the mitochondrially encoded hydrophobic subunit 6 was not present.